Dann Lab Presentation -Kayla

Thursday, April 15th, 2010

• How does manipulation of this arrangement

effect “stemness” or differentiation, OCT4, GFP, myc levels?

• Going from vector to cell line (SOHLH1)

• GFP analysis of cell lines (including TEX17)

• Immunostaining showing sub-cellular localization in COS cells

• Immunostaining analysis of cell lines: myc, and OCT4

Creating a cell line from a vector

• Plasmid backbone + insert (desired DNA sequence)

-Digest, CIP, ligate

-Gel electrophoresis for expected size purification, extract

• Bacterial transformation-Tina’s Homemade Fusion-Blue Competent Cells

*competent cells can take up the plasmid DNA

*bacterial cells in Ca2+ (permeable) mixed with plasmid DNA on

ice, heat shocked, plated (Amp)

• Mini preps for multiple colonies

• Restriction digest, gel electrophoresis

• Transfection into COS or 293

• Analyze by immunostaining or FACS

Creating pLSOGFS (SOHLH1-GFP)

• SOHLH1 - spermatogenesis and oogenesis specific basic helix-loop-helix 1 – Found in both male and female germ cells

– Essential for differentiation

• Replace Ubc promoter in Ubc-GFP– Increase differentiation activity

• SOHLH1 insert Ligation pLLU2G vector– Both cut w/ Xba1 and Age1

• Transformation

Nhe1 Age1/Xba1

Control vector – pLLU2G #3

Control vector – pLLU2G #3

8 kb

4 kb

.5 kb

1 12 23 34 45 5

Expected Age1/Xba1 band @ 4.2 kb

SOHLH1 digests following mini preps of colonies 1-5

• #1, 2, and 5 were sequenced• BLAST-ed sequencing results

• Test transfection for FACS- Increased SOHLH1 = decreased GFP

• According to FACS analysis and results of gel pLSOGFS #1 chosen for subsequent transformation and maxi prep

• Ex. of later use: test if Oct4 overexpression inhibits SOHLH1 expression (and therefore differentiation) by binding to the SOHLH1 promoter and repressing its’ transcription

Analyzing GFP reporter - FACS

GFP Analysis of Cell Lines

• 2 methods I use:– FACS (flow cytrometry)

– Immunostaining

GFP Analysis of Cell Lines

• SOGFS (SOHLH1)– Described earlier

FACS

GFP Analysis of Cell Lines• TEX17 – from ES cell mmlv,

ligated to pCDNA Not1/Xho1 (added myc), cut at Age1, ligated to pLUTG Age/CIP

Immunostaining : anti-myc + mouse IgG

pLLU2G – positive control

pLUTG-Tex17 pLUTG-WTOct #3

pCDNA-Tex17 negative control displayed no visible

GFP

Transient transfection in COS7

FACSLentivirus transduction in

DGC1

95% positive

GFP Analysis of Cell Lines

• WTO1-5• SMO1-5• NSO1-5

• Several staining experiments to look at Oct-myc &GFP using anti-GFP, anti-Oct4, anti-myc– Results –

• in most cases each cell line had some GFP visible, weak in WT, bright in NSO & SMO; a lot of background

• Myc staining has been fairly successful in 293 cells but not in germ cells (need more antibody or just doesn’t work??)

• Oct4 has seemed cytoplasmic instead of nuclear

Lentivirus transduction in DGC1 of OctWT, OctNLS,

OctSUMO/ sort

GFP Analysis of Oct Cell Lines con’t

• RNA isolation to test mRNA levels in qPCR*– mRNA levels matched FACS GFP levels – Suggests resorting GFP positives could increase Oct4

levels @ mRNA level• Resorted into high/medium– Staining: WTO not as high in Oct4 or GFP as other

lines, but in general cell lines w/ higher Oct4 exhibit higher levels of GFP

– FACS: avg % pos. WTO(6&7) = ~45 NSO(6&7) = ~85

SMO(6&7) = ~89.5

Immunostaining showing sub-cellular localization in COS cells

pCDNA-Tex17 (showing myc)

DAPI Rhodamine